See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/11848733

Candida albicans proteinases: Resolving the

mystery of a gene family

Article in Microbiology · September 2001

DOI: 10.1099/00221287-147-8-1997 · Source: PubMed

CITATIONS READS

200 69

2 authors:

Bernhard Hube Julian R Naglik

Leibniz Institute for Natural Product Research … King's College London
480 PUBLICATIONS 12,435 CITATIONS 74 PUBLICATIONS 3,825 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Candida pathogenesis View project

Experimental evolution of pathogenic fungi View project

All content following this page was uploaded by Bernhard Hube on 10 February 2014.

The user has requested enhancement of the downloaded file.

Microbiology (2001), 147, 1997–2005
Candida albicans proteinases : resolving the
REVIEW mystery of a gene family
ARTICLE
Bernhard Hube1 and Julian Naglik2
Author for correspondence : Bernhard Hube. Tel : j49 30 4547 2116. Fax : j49 30 4547 2328.
e-mail : HubeB!rki.de
1
Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany
2
Division of Oral Medicine, Pathology, Microbiology and Immunology, King’s College London
(Guy’s Campus), London, UK
Keywords : SAP, virulence factors, fungal infections
Overview
Fungal infections of mucosal surfaces are extremely
common, debilitating and often recurring diseases,
which are frequently caused by the yeast Candida
albicans. Furthermore, in the severely immuno-
compromised host, C. albicans may also cause deep-
seated or even life-threatening systemic infections. In
order to colonize, infect and evade host defence mech-
anisms, C. albicans possesses a repertoire of virulence
attributes. In particular, the secreted aspartic protein-
ases (Saps), encoded by the SAP gene family with ten
members, appear to play a major role in C. albicans
virulence. The SAP family is differentially regulated and
distinct members are expressed under a variety of
laboratory growth conditions and during experimental
C. albicans infections in vitro and in vivo. The con-
tribution of the Saps to C. albicans pathogenesis has
been clearly demonstrated using SAP-deficient mutants
and proteinase inhibitors. These studies demonstrated
that different SAP genes appear to be crucial for mucosal
and systemic infections, and are involved in C. albicans
adherence, tissue damage and evasion of host immune
responses. Therefore, the Sap isoenzymes appear to
have a variety of functions in vivo, which are probably
called upon at different stages and in different types of C.
albicans infection. This review aims to summarize the
more recent data regarding the contribution of the
secreted proteinases to C. albicans virulence and strives
to explain why C. albicans possesses such a gene family.
Opportunistic fungal infections : from
commensal to pathogen
C. albicans is a ubiquitous mucosal commensal yeast
that is commonly isolated from the oral cavity, the
gastro-intestinal tract and the vagina, where it resides in
equilibrium with the microbial flora and the host
immune system. The physiological status of the host is
0002-4881 # 2001 SGM
Printed in Great Britain
the primary factor governing the aetiology of candidosis.
However, the observation that only slight alterations in
this physiological state can turn normally harmless
commensal yeasts into aggressive pathogens causing
mucosal, superficial or even life-threatening systemic
infections in the immunocompromised host points to
the pathogenic potential of Candida species (Fidel et al.,
1999). An increasing number of immunocompromised
individuals, such as those with HIV infection, neutro-
paenia, intensive care patients and those treated with
immunosuppressive drugs after organ transplantation,
experience some form of mucosal Candida infection. In
addition, nearly three-quarters of all healthy women
experience at least one vaginal yeast infection (Sobel,
1985). Furthermore, while the high mortality rate
associated with systemic bacterial infections has
declined with the early administration of broad-spec-
trum antibiotics, systemic fungal infections have become
increasingly significant in causing morbidity and mor-
tality in immunocompromised patients (Fidel et al.,
1999). As a result, Candida has become the fourth most
common nosocomial bloodstream pathogen in the USA
(Jarvis, 1995). Yet despite the growing importance of
Candida infections in medicine, many of its interactions
with the host are still poorly understood.
Virulence attributes of C. albicans
The process, development and course of microbial
infections may be regarded as an encounter between the
virulence of a micro-organism and the ability of the host
to prevent or resist microbial colonization or invasion.
Pathogenic bacteria, having a relatively small genome,
often develop very specific ways of causing host
infections. For example, several bacteria have developed
highly specialized virulence factors, which are adapted
to intracellular survival within a specific host cell type,
and in a few cases single toxins may cause disease.
Conversely, C. albicans is not a prokaryote but a
1997
B. H U B E a n d J. N A G L I K

Sap4 nized as the most credible (Odds, 1994). Extracellular

proteolytic activity had already been discovered in the
Sap6 mid-sixties (Staib, 1965) but it was not until the early
nineties, when molecular methods were introduced into
Sap5 the Candida field, that scientists began to understand
the genetic complexity of this fungus. For instance, a
gene (SAP1) encoding an extracellular proteinase was
Sap1 cloned in 1991 and was thought to be responsible for the
observed secretory aspartic proteinase (Sap) activity of
Sap2 C. albicans (Hube et al., 1991). However, a detailed
study of the C. albicans genome in recent years indicated
Sap3 that SAP1 was just the tip of an iceberg and revealed that
the fungus possesses an arsenal of ten SAP genes that
encode extracellular proteinases. The fact that C.
Sap8 albicans can encounter a large number of different
tissues during the infection process in vivo may provide
clues as to why it has evolved to possess such a large
repertoire of SAP genes.
Sap9

Sap10
The SAP gene family

Sap7 Shortly after SAP1 was discovered, more genes encoding

extracellular aspartic proteinases were identified, by
................................................................................................................................................. PCR-based cloning strategies (Wright et al., 1992), by
Fig. 1. Dendrogram of the C. albicans Sap isoenzyme family using SAP1 as a probe (Monod et al., 1994, 1998 ; White
based on amino acid sequences. Three distinct groups are
clustered within the family. Sap1–3 are up to 67 % and Sap4–6
et al., 1993), by sequencing the promoter region of SAP1
up to 89 % identical, while Sap7 is only 20–27 % identical to (Miyasaki et al., 1994) or by  searches and sequence
other Saps. Sap9 and Sap10 both have C-terminal consensus alignments in the Candida genome databases (e.g.
sequences typical for GPI proteins. Reprinted from Stehr et al. SAP10, accession number AF146440). The deduced
(2000) with permission of the publisher. proteins are all aspartic proteinases and share a number
of Sap-specific characteristics (Fig. 1). All ten SAP genes
encode preproenzymes approximately 60–200 amino
acids longer than the mature enzyme. The N-terminal
eukaryote, with a larger genome and thus greater secretion signal is cleaved by a signal peptidase in the
resources. Accordingly, C. albicans, although highly endoplasmic reticulum (ER) (Fig. 2). The propeptide is
adapted to humans as a commensal organism, possesses removed to activate the proteinases by the subtilisin-like
a larger repertoire of virulence attributes in order to Kex2 proteinase in the Golgi before being transported
colonize the host, and inflict damage directly or in- via vesicles to the cell surface for secretion or glycosyl-
activate, resist or misdirect host defence mechanisms. phosphatidylinositol (GPI)-anchoring. Although Kex2
However, in principle, the main aim of a micro- may be a key regulatory proteinase of Saps (Newport &
organism is not to cause death or even damage the host Agabian, 1997), other alternative processing pathways
but to survive and reproduce, during which damage may are thought to exist (Togni et al., 1996), and auto-
only be a side effect. Indeed, in C. albicans it appears activation was shown to occur extracellularly for Sap1–3
that the transition from harmless commensal to disease- and Sap6 at certain pH values (Koelsch et al., 2000). The
causing pathogen is finely balanced, and is attributable mature enzyme contains sequence motifs typical for all
to an extensive repertoire of virulence determinants aspartic proteinases, including the two conserved
selectively expressed under suitable predisposing con- aspartate residues of the active site. Conserved cysteine
ditions. residues are probably implicated in maintaining the
three-dimensional structure of the enzyme (Hube, 1996).
The virulence factors expressed or required by C. Unlike Sap1–8, Sap9 and Sap10 both have C-terminal
albicans to cause infections may well vary, depending on consensus sequences typical for GPI proteins (Fig. 2).
the type of infection, the stage and site of infection, and
the nature of the host response. Thus, C. albicans must While in the early nineties efforts concentrated on the
be highly adapted to an existence on and within the host, collection of sequence data, the increasing numbers of
which indicates that this fungus possesses virulence cloned SAP genes soon shifted the interest towards the
attributes distinct from those of the closely related, role and function of the SAP gene family during the
but non-pathogenic yeast Saccharomyces cerevisiae. infection process. The fact that the presence of a SAP
Although a number of potential virulence factors have gene family was unique to only the most pathogenic
been suggested for C. albicans, cell morphology, ad- Candida species, such as C. albicans (Magee et al., 1993,
hesion factors, phenotypic switching and extracellular 1994), C. dubliniensis (Gilfillan et al., 1998), C. tropicalis
lipolytic or proteolytic activity have long been recog- (Zaugg et al., 2001) and C. parapsilosis (Monod et al.,

1998
 Candida SAP gene family

Microniche
in host
CM CW

ER Golgi
Sap1–3

pH 2–5

GPI?
Sap9–10
GPI?
Signal
peptidase Kex2

pH 3–7

Sap4–6

Sap7, 8?
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Processing and secretion of Saps via the secretory pathway and their optimal pH activity ranges. SAP genes are
translated as preproprotein into the ER. The signal peptide is processed by the signal peptidase (arrow) in the ER. The
propeptide contains two Lys-Arg processing sites, which are targeted by the proteinase Kex2 (arrow) in the Golgi
apparatus or by alternative activation processes (see text). The propeptide was found to be essential for proper folding
of Saps (Beggah et al., 2000). The mature Sap proteins are transported by vesicles via the cell membrane into the cell wall
and secreted into the extracellular space. Sap1–3 are mostly active between pH 2 and 5 while Sap4–6 are active at a
higher pH range (3–7) and thus may act in different host environments. Sequence analysis of Sap9 and Sap10 suggested
that both proteinases are GPI-proteins anchored in the cell membrane or cell wall (Caro et al., 1997).

1994), but was absent in the non-pathogenic yeast S. structures and intercellular substances, or by destroying
cerevisiae, supported the view that these proteinases cells and molecules of the host immune system to avoid
may be involved in virulence. However, in the light of or resist antimicrobial attack. Such activities for Sap2
the discovery that only a single isoenzyme, Sap2, was have been shown in vitro (reviewed by Ru$ chel, 1992 ; and
necessary and sufficient to allow rapid growth of C. Hube, 1996, 1998). Extracellular matrix and host surface
albicans in media containing protein as the sole source proteins such as keratin, collagen, laminin, fibronectin
of nitrogen (Hube et al., 1994 ; White & Agabian, 1995), and mucin are efficiently degraded by Sap2. Several host
it remained a mystery as to why this fungus possessed a defence proteins, such as salivary lactoferrin, the pro-
whole family of SAP genes. As one possible explanation teinase inhibitor α-macroglobulin, enzymes of the res-
may be that different proteinases are required to act piratory burst of macrophages and almost all immuno-
upon different host proteins and tissues in vivo, a globulins, including secretory IgA, which is normally
number of studies dealt with the possible targets of Saps. resistant to most bacterial proteinases, can also be
hydrolysed by Sap2.
Possible target proteins of C. albicans C. albicans Saps may also act on host proteolytic
proteinases in vivo cascades with numerous effects, which have no obvious
advantage for the fungus. For example, Sap2 can activate
At the most basic level, one role of the C. albicans Saps host protein precursors of the blood clotting cascade
may be to simply digest host proteins to provide nitrogen (Kaminishi et al., 1994), inactivate the epidermal
for the cells. Since Sap2 activity enables the fungus to cysteine proteinase inhibitor cystatin A (Tsushima et al.,
grow efficiently in media containing serum albumin or 1994), and cleave human endothelin-1 precursor (a
other proteins as a sole source of nitrogen, nutrient vasoconstrictive peptide) to alter vascular homeostasis
acquisition is a likely function of the proteinases. (Tsushima & Mine, 1995). Such activities may be
However, the Saps may have also adapted and evolved responsible for the enhanced overall circulating pro-
to have more direct virulence functions. For example, teolytic activity observed in traumatized mice challenged
Saps could contribute to host tissue adhesion and with C. albicans (Neely et al., 1994). Furthermore,
invasion by degrading or distorting host cell surface Candida proteinases have been shown to activate the

1999
B. H U B E a n d J. N A G L I K
proinflammatory cytokine interleukin-1β from its pre-
cursor, suggesting a role for Saps in the activation and
maintenance of the inflammatory response at epithelial
surfaces in vivo (Beausejour et al., 1998).
These studies suggested that Sap2, in contrast to the
highly substrate-specific enzymes produced by certain
bacteria, has very broad substrate specificity and may
have multiple targets in vivo. However, this raises the
question of why, if a single proteinase has such a range
of activities and functions, does C. albicans need a
family of ten SAP genes ? Magee et al. (1993) postulated
that the Sap isoenzymes might have a variety of functions
in vivo, which may be called upon at different sites, and
during different stages and types of C. albicans infection.
SAP gene expression in vitro and in vivo
The presence of ten SAP genes indicated that different
proteinases might target a variety of host cells and
tissues during the infection process. If this were the case,
one would expect the various members of the SAP
family to be differentially regulated and expressed under
a variety of laboratory growth conditions and during C.
albicans infections in vivo. Accordingly, a number of
studies have addressed this issue (Hube, 2000).
Under most proteinase-inducing conditions in the lab-
oratory, the major proteinase gene expressed in C.
albicans yeast forms is SAP2, which was found to be
regulated by a positive feedback mechanism : peptides
resulting from proteolysis of high-molecular-mass
proteins were proposed to lead to the induction of SAP2
gene expression (Hube et al., 1994). In contrast, SAP1
and SAP3 were discovered to be differentially expressed
during phenotypic switching in certain strains (Morrow
et al., 1992 ; White et al., 1993). However, later studies
indicated the regulation of SAP3 during switching was
not absolute (Hube et al., 1994 ; White & Agabian,
1995 ; Smolenski et al., 1997). Expression of SAP8 is
temperature-regulated (Monod et al., 1998) and SAP9
and SAP10 are constitutively expressed under most
environmental conditions in both yeast and hyphal
forms (A. Felk, W. Scha$ fer & B. Hube, unpublished
results). Since most aspartic proteinases are only active
under acidic conditions, it was perhaps a surprising
discovery that the SAP4–6 genes were almost exclusively
expressed during hyphal formation at neutral pH, even
in defined protein-free media (Hube et al., 1994 ; White
& Agabian, 1995). These studies demonstrated that the
SAP gene family was differentially expressed in vitro and
further suggested that, in contrast to the induction of
SAP2, expression of other SAP genes was not dependent
on the presence of exogenous protein or peptides.
The in vitro demonstration of distinct SAP expression
patterns in yeast, hyphal and phenotypically switched
cells indicated that proteinase expression was a highly
regulated and tightly controlled process. However, this
did not address the issue of whether the Sap family
contributed to the pathogenicity of C. albicans in vivo.
For this reason it was crucial to first ascertain whether
2000
the proteinases were also differentially expressed during
C. albicans infections ; this was demonstrated using in
vitro and animal infection experimental models.
In vitro experimental models of oral (Schaller et al.,
1998) and cutaneous (Schaller et al., 2000) C. albicans
infections suggested that SAP1–3 were the main
proteinases expressed during superficial infections. This
supposition was supported by the detection of SAP1 and
SAP2 transcripts in a rat vaginitis model (De Bernardis
et al., 1995). In contrast to mucosal infection models,
experimental models of systemic C. albicans infections
correlated SAP4–6 expression with systemic disease (A.
Felk and others, unpublished results ; Staib et al., 2000).
However, using in vivo expression technology, Staib et
al. (1999) also demonstrated SAP2 expression in late
stages of systemic infections. Taken together, these in
vitro experimental and animal model data correlated
SAP gene expression with C. albicans virulence and
demonstrated differential SAP gene expression during
different types of C. albicans infection.
Whether these models were representative of proteinase
expression during human mucosal and systemic
infections was not known. However, high titres of anti-
Sap antibodies have been observed in sera of candidosis
patients, indicating the presence of Sap antigen during
infection (Ru$ chel, 1992). In addition, Sap antigens have
been detected in biopsies of oral epithelial lesions
collected from HIV-infected patients (Schaller et al.,
1999a) and in almost all organs of immunocompromised
patients who had died of systemic C. albicans infections
(Ru$ chel et al., 1991). Nevertheless, only two studies
have investigated the expression of the SAP gene family
during human C. albicans infections. Schaller et al.
(1998) showed that the SAP genes were indeed expressed
during oral candidosis. Moreover, Naglik et al. (1999)
and J. R. Naglik and others (unpublished results) were
able to demonstrate differential expression of the SAP
gene family, with SAP1, SAP3 and SAP7 transcripts
predominantly being expressed in patients with oral C.
albicans infection as opposed to oral C. albicans carriers.
Therefore, differential expression of the proteinase
family has been demonstrated in culture media, during
in vitro and animal experimental infection models and
during human C. albicans infections. Such differential
expression suggested a more specific role or function for
the different SAP genes. However, although these studies
correlated SAP gene expression with the ability of C.
albicans to cause infection, they did not directly dem-
onstrate that Sap proteinases actually contributed to the
pathology or virulence of C. albicans infections.
Contribution of Saps to the pathogenesis of
C. albicans infections
Strong evidence existed to indicate that Candida
proteinases were differentially expressed in vivo and had
the potential to degrade a large number of host proteins,
but their direct contribution to pathogenesis was still
unknown. However, recent studies using proteinase
 Candida SAP gene family

Table 1. Use of SAP-deficient mutants to determine a role for the proteinases during
C. albicans infections

Study Main findings

Mucosal infections
Watts et al. (1998) sap1, sap2 and sap3 null mutants were less adherent to buccal
epithelial cells than the parental strain. The sap4–6 triple mutant
showed significantly increased adherence.
Schaller et al. (1999b) sap1, sap2 and sap3 mutants caused less tissue damage than the
parental strain. A sap1\3 double mutant caused fewer lesions than
the two sap1 or sap3 mutants individually. sap4–6 mutant showed
equal or enhanced tissue damage.
De Bernardis et al. (1999) sap1, sap2 and sap3 mutants, but not the sap4–6 mutant, were less
virulent in a rat vaginitis model compared with the parent strain.
The sap2 mutant was almost avirulent.
Systemic infections
Ibrahim et al. (1998) No difference in adherence to endothelial cells between the sap1,
sap2 and sap3 null mutants and the parental strain. The sap2
mutant caused less damage to endothelial cells.
Kretschmar et al. (1999) The sap4–6 mutant, but not sap1, sap2 or sap3 mutants, caused less
tissue damage and invasion in peritoneal infections.
Hube et al. (1997) ; sap1, sap2, sap3 and, in particular, sap4–6 mutants were less lethal
Sanglard et al. (1997) in two animal models compared with the parent strain. The sap4–6
mutant displayed the greatest attenuation.
Interactions with immune system
Borg-von Zepelin et al. The sap4–6 triple mutant was eliminated 53 % more effectively after
(1998) phagocytosis by macrophages than the parent strain.

inhibitors and SAP-disrupted or SAP-overexpressing
mutants have compellingly demonstrated that Saps
indeed contribute to the virulence of C. albicans.
Inhibition of Saps with the aspartic proteinase inhibitor
pepstatin A prevented the initial penetration of C.
albicans through mucosal surfaces, but not the dis-
semination of the fungus once the cells had already
reached the blood vessels (Fallon et al., 1997). Moreover,
pepstatin A prevented C. albicans invading and causing
tissue damage in oral, vaginal and skin experimental
infection models (Schaller et al., 1999b ; De Bernardis
et al., 1997). In addition, HIV aspartic proteinase
inhibitors, which also act on the C. albicans Saps
(Gruber et al., 1999 ; Borg-von Zepelin et al., 1999 ;
Cassone et al., 1999 ; Korting et al., 1999), can inhibit C.
albicans adherence to epithelial cells (Borg-von Zepelin
et al., 1999). Taken together, these data indicate that Sap
activity may be crucial during the early stages of C.
albicans infections.
Although Sap2 was the dominant C. albicans extra-
cellular proteinase in vitro, the in vivo expression
pattern of SAP genes suggested that Sap2 was not the
only proteinase and certainly not the most dominant
proteinase acting in vivo. Therefore, to determine to
what extent the different Saps contributed to the
different types of Candida infection, mutants lacking
(Table 1) or overexpressing (Dubois et al., 1998 ; Kvaal
et al., 1999) distinct SAP genes were created and used in
experimental infections. These studies showed that not
only were distinct Sap isoenzymes required during
different types of infection, but also that not all Candida
proteinases were important for virulence per se (Dubois
et al., 1998). Using SAP-deficient mutants, Schaller et al.
(1999b) demonstrated that SAP1–3, but not SAP4–6,
contributed significantly to experimental C. albicans
infections of artificial oral mucosa. A major role for
SAP1–3, but not SAP4–6, was also demonstrated in
experimental vaginal infections using SAP-disrupted
mutants (DeBernardis et al., 1999). Using a gene
misexpression strategy in the switching strain WO-1 in
which white-phase cells misexpressed the opaque-
specific gene SAP1, Kvaal et al. (1999) demonstrated in a
cutaneous mouse model that SAP1 caused a dramatic
increase in cutaneous infection, probably as a result of
increased adherence to, and ‘ cavitation ’ of, the skin.
In contrast, SAP4–6, but not SAP1–3, appeared to be
critical for systemic infections, as mutants lacking
SAP4–6 were strongly attenuated in two rodent models
of intravenous infection (Hube et al., 1997 ; Sanglard
et al., 1997) and during intraperitoneal infection
(Kretschmar et al., 1999) (Table 1). In this model,
SAP4–6 may contribute to virulence by resisting phago-
cytic attack, since the corresponding proteinases were
mostly expressed by C. albicans in phagolysosomes of
murine peritoneal macrophages and neutrophils (Borg-
von Zepelin et al., 1998 ; A. Felk and others, unpublished

2001
B. H U B E a n d J. N A G L I K
results). Furthermore, SAP4–6-deficient mutants were
shown to be hypersensitive to phagocytic killing by
macrophages (Borg-von Zepelin et al., 1998).
These data clearly demonstrate that Saps contribute to
the pathogenesis of Candida infections. However, it
should be noted that mutants lacking individual SAP
genes rarely exhibit a full avirulent phenotype in a
particular infection model. This may indicate a syn-
ergistic effect of a number of proteinases during certain
infections, or the involvement of other virulence factors.
Nonetheless, these studies illustrate that not all of the
proteinases are required at the same time or stage of the
infection process and not all contribute to the same
types of infection. Determining the precise roles and
functions of the C. albicans proteinases in vivo should
provide a big step forward in identifying which
proteinases, or set of proteinases, may contribute to, or
even be responsible for, specific types of C. albicans
infection.
Why does C. albicans possess a SAP gene
family ?
There are several possible reasons or circumstances that
would make the assembly of a family of proteinases
necessary for C. albicans.
Firstly, the different Sap isoenzymes may have adapted
and evolved to function in different tissues and environ-
ments. Such a view may also explain how the gene
family evolved : C. albicans may have duplicated a
successful factor (an ancestral proteinase), which in turn
adapted to different host environments and thus evolved
into homologous but functionally distinct Sap iso-
enzymes. This hypothesis may be supported by the fact
that all the SAP genes encode similar amino acid
sequences (Fig. 1). However, since only SAP1 and SAP4
are clustered in tandem and all other SAP genes are
located on five different chromosomes, this gene dupli-
cation event probably occurred sometime in the distant
past. Alternatively, it is possible that the high genetic
flexibility of C. albicans may have accelerated this
widespread distribution of the SAP genes.
Although the overall similarity of the SAP genes is high,
differences in their promoter sequences indicate that
expression of the various SAP genes is controlled by
different SAP-specific transcriptional regulators. This
would suggest that the SAP genes might have evolved to
possess distinct properties and functions, which indeed
appears to be the case. For example, different Saps have
been shown to have different pH optima for activity
(Borg-von Zepelin et al., 1998). Sap2 acts mainly at
acidic pH values around pH 4n0, Sap4–6 are significantly
active at physiological pH and Sap3 still has activity at
pH 2n0. This provides C. albicans with a range of
proteolytic activity from pH 2n0 to 7n0, a property that
may prove essential for the specific adaptation of
individual Saps to different host environments.
It seems surprising that all secreted proteinases of C.
albicans are aspartic proteinases and that neither extra-
2002
cellular serine-, metallo- nor cysteine proteinases have
been identified in pathogenic strains of C. albicans. In
contrast, other human pathogenic fungi, such as the
filamentous fungus Aspergillus fumigatus, secrete sev-
eral classes of different proteinases, including aspartic,
serine and metalloproteinases, although none of these
enzymes has been definitively associated with virulence
(Monod et al., 1999). This may disadvantage C. albicans
since aspartic proteinases (with a few exceptions, such
as human renin) are usually only active in acidic pH
ranges. However, this may be compensated for not only
by the adaptation of particular Sap isoenzymes to
function at higher pH values, but also by the ability of C.
albicans to actively acidify its surrounding environment
to provide microniches optimal for Sap activity during
infection. These properties may be essential for the
fungus when infecting the vaginal mucosa or the oral
cavity.
Secondly, the C. albicans SAP gene family may have
evolved to allow coordinated regulation of the pro-
teinases with other virulence factors. This may not only
explain why certain SAP genes are induced in the absence
of exogenous protein or peptides, but also the differences
between the SAP promoter sequences. Key trans-
criptional factors would permit the expression of specific
Saps when C. albicans needs to call upon other virulence
factors or when it encounters different host environ-
ments. Therefore, the same transcriptional factor may
regulate several genes to allow the simultaneous ex-
pression of a combination of virulence factors to respond
to local environmental challenges. For example, SAP4–6
were found to be regulated during the yeast-to-hyphal
transition, which in turn is known to be regulated by key
transcriptional factors such as Efg1 (Ernst, 2000). It is
currently not precisely known why mutants lacking
EFG1 are avirulent in several infection models. How-
ever, it seems that Efg1 is a global regulator not only of
hyphal formation, but also of a number of other
virulence attributes, including proteinase expression
(Schro$ ppel et al., 2000). A lack of this factor would
subsequently cause severe defects in the virulence
potential of C. albicans. In addition, SAP1 and SAP3
were shown to be regulated during phenotypic switching
in particular strains, which involves the rapid change of
a large number of different phenotypes and is pre-
sumably regulated via a master switch mechanism (Soll,
1997). This specific coordinated regulation of the SAP
gene family with various virulence factors indicates that
the proteinases may have evolved to specifically enhance
the pathogenic ability of C. albicans.
A third possible reason for a family of proteinase genes
is that the concomitant expression of a number of
similar but functionally distinct SAP genes, rather than
the expression of a single SAP gene, may result in a
synergistic effect to promote colonization or infection.
Thus, several Saps may act in unison to carry out a series
of tasks to possibly provide C. albicans with a biological
advantage.
Finally, providing an arsenal of SAP genes has the
advantage of having a second enzyme in line when one

fails (i.e. the enzyme may be functionally redundant), is
removed or is otherwise lost. There is evidence for such
compensatory mechanisms in C. albicans. For instance,
disruption of SAP1 and SAP3 genes, which may be
required for oral infection, lead to increased expression
of SAP5 and SAP8, suggesting that C. albicans may be
attempting to compensate for the loss of key genes by
upregulating alternative genes (Schaller et al., 1999b).
Such balanced regulation is likely to be a general
phenomenon in C. albicans.
Conclusion and future directions
The presence of a SAP gene family in C. albicans clearly
provides the fungus with an efficient and flexible
proteolytic system that may prove vital to its success
as an opportunistic pathogen. Furthermore, Sap pro-
duction is a highly regulated and tightly controlled
process, which appears to be a central factor in many
aspects of C. albicans virulence and is indicative of the
multiple functions this gene family possesses. These
include the simple role of digesting molecules for
nutrient acquisition, the contribution to host tissue
invasion by digesting or distorting host cell membranes,
the degradation of host surface molecules to enhance
adhesion, and the digestion of cells and molecules of the
host immune system to avoid or to resist antimicrobial
attack.
However, there are still many unanswered questions
that need to be addressed. (1) How is proteinase
expression regulated ? The signal transduction pathways
that control the differential expression of SAP genes are
currently unknown and no receptor has yet been
implicated in the regulation of C. albicans proteinase
secretion. (2) What are the actual targets of Saps during
human infections ? Although the possible targets of the
proteinases have been deduced from in vitro studies,
these need to be confirmed in the in vivo environment.
Furthermore, as the substrate specificity of the Saps is so
broad and most Sap antigen is detected within the C.
albicans cell wall (Ru$ chel et al., 1991 ; Schaller et al.,
1999a), how does the fungus manage to protect itself
from proteolytic digestion ? (3) The precise roles and
functions of the SAP genes, especially SAP7, SAP9 and
SAP10, during human infections are currently unknown,
as is the reason why Sap9 and Sap10 have C-terminal
GPI-anchoring sequences. Do these two proteinases
have functions similar to the GPI-anchored yapsins of S.
cerevisiae (Komano et al., 1999) or do they contribute to
C. albicans infection ? (4) The immunological conse-
quences of Sap secretion and other interactions of the
proteinases with host factors in vivo are largely un-
known and need to be addressed.
In summary, this review has endeavoured to elucidate
the possible reasons why C. albicans is equipped with a
family of proteinase genes and suggests a number of
possible explanations for their evolution in light of the
information available. However, there are several short-
falls that need to be tackled before the biological
significance of this gene family is fully understood. With
Candida SAP gene family
access to the complete genome sequence of C. albicans
and with the development of new and exciting tools
such as DNA microarray analysis, our understanding of
the SAP gene family and its interaction with the human
host should rapidly increase.
Acknowledgements
We apologise for omissions resulting from space limitations.
Our own work is supported by the Deutsche Forschungs-
gemeinschaft (Hu 528\7 and Hu 525\8), the European
Commission (‘ Galar Fungail ’) and the Dunhill Medical
Research Trust. We thank Neil A. R. Gow, University of
Aberdeen, UK, for critical reading of the manuscript.
References
Beausejour, A., Grenier, D., Goulet, J. P. & Deslauriers, N. (1998).
Proteolytic activation of the interleukin-1β precursor by Candida
albicans. Infect Immun 66, 676–681.
Beggah, S., Lechenne, B., Reichard, U., Foundling, S. & Monod, M.
(2000). Intra- and intermolecular events direct the propeptide-
mediated maturation of the Candida albicans secreted aspartic
proteinase Sap1p. Microbiology 146, 2765–2773.
Borg-von Zepelin, M., Beggah, S., Boggian, K., Sanglard, D. &
Monod, M. (1998). The expression of the secreted aspartic
proteinases Sap4 to Sap6 from Candida albicans in murine
macrophages. Mol Microbiol 28, 543–554.
Borg-von Zepelin, M., Meyer, I., Thomssen, R., Wu$ rzner, R.,
Sanglard, D., Telenti, A. & Monod, M. (1999). HIV-protease
inhibitors reduce cell adherence of Candida albicans strains by
inhibition of yeast secreted aspartic proteases. J Investig Dermatol
113, 747–751.
Caro, L. H., Tettelin, H., Vossen, J. H., Ram, A. F., van den Ende, H.
& Klis, F. M. (1997). In silicio identification of glycosyl-
phosphatidylinositol-anchored plasma-membrane and cell wall
proteins of Saccharomyces cerevisiae. Yeast 13, 1477–1489.
Cassone, A., De Bernardis, F., Torosantucci, A., Tacconelli, E.,
Tumbarello, M. & Cauda, R. (1999). In vitro and in vivo
anticandidal activity of human immunodeficiency virus protease
inhibitors. J Infect Dis 180, 448–453.
De Bernardis, F., Cassone, A., Sturtevant, J. & Calderone, R.
(1995). Expression of Candida albicans SAP1 and SAP2 in
experimental vaginitis. Infect Immun 63, 1887–1892.
De Bernardis, F., Boccanera, M., Adriani, D., Spreghini, E.,
Santoni, G. & Cassone, A. (1997). Protective role of antimannan
and anti-aspartic proteinase antibodies in an experimental model
of Candida albicans vaginitis in rats. Infect Immun 65, 3399–3405.
De Bernardis, F., Arancia, S., Morelli, L., Hube, B., Sanglard, D.,
Scha$ fer, W. & Cassone, A. (1999). Evidence that members of the
secretory aspartic proteinase gene family, in particular SAP2, are
virulence factors for Candida vaginitis. J Infect Dis 179, 201–208.
Dubois, N., Colina, A. R., Aumont, F., Belhumeur, P. & de
Repentigny, L. (1998). Overexpression of Candida albicans
secretory aspartic proteinase 2 and its expression in Saccharo-
myces cerevisiae do not augment virulence in mice. Microbiology
144, 2299–2310.
Ernst, J. F. (2000). Transcription factors in Candida albicans –
environmental control of morphogenesis. Microbiology 146,
1763–1774.
Fallon, K., Bausch, K., Noonan, J., Huguenel, E. & Tamburini, P.
(1997). Role of aspartic proteases in disseminated Candida
albicans infection in mice. Infect Immun 65, 551–556.
2003
B. H U B E a n d J. N A G L I K
Fidel, P. L., Vazquez, J. A. & Sobel, J. D. (1999). Candida glabrata :
review of epidemiology, pathogenesis, and clinical disease with
comparison to C. albicans. Clin Microbiol Rev 12, 80–96.
Gilfillan, G. D., Sullivan, D. J., Haynes, K., Parkinson, T., Coleman,
D. C. & Gow, N. A. (1998). Candida dubliniensis : phylogeny and
putative virulence factors. Microbiology 144, 829–838.
Gruber, A., Berlit, J., Speth, C., Lass-Florl, C., Kofler, G., Nagl, M.,
Borg-von Zepelin, M., Dierich, M. P. & Wu$ rzner, R. (1999).
Dissimilar attenuation of Candida albicans virulence properties
by human immunodeficiency virus type 1 protease inhibitors.
Immunobiology 201, 133–144.
Hube, B. (1996). Candida albicans secreted aspartic proteinases.
Curr Top Med Mycol 7, 55–69.
Hube, B. (1998). Possible role of proteinases in Candida infections.
Rev Iberoam Micol 15, 68–71.
Hube, B. (2000). Extracellular proteinases of human pathogenic
fungi. Contrib Microbiol 5, 126–137.
Hube, B., Turver, C. J., Odds, F. C., Eiffert, H., Boulnois, G. J.,
Ko$ chel, H. & Ru$ chel, R. (1991). Sequence of the Candida albicans
gene encoding the secretory aspartate proteinase. J Med Vet
Mycol 29, 129–132.
Hube, B., Monod, M., Schofield, D. A., Brown, A. J. & Gow, N. A.
(1994). Expression of seven members of the gene family encoding
secretory aspartic proteinases in Candida albicans. Mol Microbiol
14, 87–99.
Hube, B., Sanglard, D., Odds, F. C., Hess, D., Monod, M., Scha$ fer,
W., Brown, A. J. & Gow, N. A. (1997). Disruption of each of the
secreted aspartic proteinase genes SAP1, SAP2, and SAP3 of
Candida albicans attenuates virulence. Infect Immun 65,
3529–3538.
Ibrahim, A. S., Filler, S. G., Sanglard, D., Edwards, J. E., Jr & Hube,
B. (1998). Secreted aspartic proteinases and interactions of
Candida albicans with human endothelial cells. Infect Immun 66,
3003–3005.
Jarvis, W. R. (1995). Epidemiology of nosocomial fungal
infections, with emphasis on Candida species. Clin Infect Dis 20,
1526–1530.
Kaminishi, H., Hamatake, H., Cho, T., Tamaki, T., Suenaga, N.,
Fujii, T., Hagihara, Y. & Maeda, H. (1994). Activation of blood
clotting factors by microbial proteinases. FEMS Microbiol Lett
121, 327–332.
Koelsch, G., Tang, J., Loy, J. A., Monod, M., Jackson, K.,
Foundling, S. I. & Lin, X. (2000). Enzymic characteristics of
secreted aspartic proteases of Candida albicans. Biochim Biophys
Acta 1480, 117–131.
Komano, H., Rockwell, N., Wang, G. T., Krafft, G. A. & Fuller,
R. S. (1999). Purification and characterization of the yeast glyco-
sylphosphatidylinositol-anchored, monobasic-specific aspartyl
protease yapsin 2 (Mkc7p). J Biol Chem 274, 24431–24437.
Korting, H. C., Schaller, M., Eder, G., Hamm, G., Bo$ hmer, U. &
Hube, B. (1999). Effects of the human immunodefiency virus
(HIV) proteinase inihibitors saquinavir and indinavir on the in
vitro activities of secreted aspartic proteinases of Candida
albicans isolates from HIV-infected patients. Antimicrob Agents
Chemother 43, 2038–2042.
Kretschmar, M., Hube, B., Bertsch, T., Sanglard, D., Merker, R.,
Schroder, M., Hof, H. & Nichterlein, T. (1999). Germ tubes and
proteinase activity contribute to virulence of Candida albicans in
murine peritonitis. Infect Immun 67, 6637–6642.
Kvaal, C., Lachke, S. A., Srikantha, T., Daniels, K., McCoy, J. & Soll,
D. R. (1999). Misexpression of the opaque-phase-specific gene
PEP1 (SAP1) in the white phase of Candida albicans confers
2004
increased virulence in a mouse model of cutaneous infection.
Infect Immun 67, 6652–6662.
Magee, B. B., Hube, B., Wright, R. J., Sullivan, P. J. & Magee, P. T.
(1993). The genes encoding the secreted aspartyl proteinases of
Candida albicans constitute a family with at least three members.
Infect Immun 618, 3240–3243.
Miyasaki, S. H., White, T. C. & Agabian, N. (1994). A fourth
secreted aspartic proteinase gene (SAP4) and a CARE2 repetitive
element are located upstream of the SAP1 gene in Candida
albicans. J Bacteriol 176, 1702–1710.
Monod, M., Togni, G., Hube, B. & Sanglard, D. (1994). Multiplicity
of genes encoding secreted aspartic proteinases in Candida
species. Mol Microbiol 13, 357–368.
Monod, M., Hube, B., Hess, D. & Sanglard, D. (1998). Differential
regulation of SAP8 and SAP9, which encode two new members of
the secreted aspartic proteinase family in Candida albicans.
Microbiology 144, 2731–2737.
Monod, M., Jaton-Ogay, K. & Reichard, U. (1999). Aspergillus
fumigatus-secreted proteases as antigenic molecules and virulence
factors. Contrib Microbiol 2, 182–192.
Morrow, B., Srikantha, T. & Soll, D. R. (1992). Transcription of the
gene for a pepsinogen, PEP1, is regulated by white-opaque
switching in Candida albicans. Mol Cell Biol 12, 2997–3005.
Naglik, J. R., Newport, G., White, T. C., Fernandes-Naglik, L. L.,
Greenspan, J. S., Greenspan, D., Sweet, S. P., Challacombe, S. J. &
Agabian, N. (1999). In vivo analysis of secreted aspartic proteinase
expression in human oral candidiasis. Infect Immun 67,
2482–2490.
Neely, A. N., Miller, R. G. & Holder, I. A. (1994). Proteolytic
activity and fatal gram-negative sepsis in burned mice : effect of
exogenous proteinase inhibition. Infect Immun 62, 2158–2164.
Newport, G. & Agabian, N. (1997). KEX2 influences Candida
albicans proteinase secretion and hyphal formation. J Biol Chem
272, 28954–28961.
Odds, F. C. (1994). Candida species and virulence. ASM News 60,
313–318.
Ru$ chel, R. (1992). Proteinase. In New Strategies in Fungal Disease,
pp. 17–31. Edited by J. E. Bennett, R. J. Hay & P. K. Peterson.
Edinburgh : Churchill Livingstone.
Ru$ chel, R., Zimmermann, F., Bo$ ning-Stutzer, B. & Helmchen, U.
(1991). Candidosis visualised by proteinase-directed immuno-
fluorescence. Virchows Arch A Pathol Anal Histopathol 419,
199–202.
Sanglard, D., Hube, B., Monod, M., Odds, F. C. & Gow, N. A.
(1997). A triple deletion of the secreted aspartic proteinase genes
SAP4, SAP5, and SAP6 of Candida albicans causes attenuated
virulence. Infect Immun 65, 3539–3546.
Schaller, M., Scha$ fer, W., Korting, H. C. & Hube, B. (1998).
Differential expression of secreted aspartic proteinases in a model
of human oral candidosis and in patient samples from the oral
cavity. Mol Microbiol 29, 605–615.
Schaller, M., Hube, B., Ollert, M. W., Scha$ fer, W., Borg-von
Zepelin, M., Thoma-Greber, E. & Korting, H. C. (1999a). In vivo
expression and localization of Candida albicans secreted aspartic
proteinases during oral candidosis in HIV-infected patients. J
Investig Dermatol 112, 383–386.
Schaller, M., Korting, H. C., Scha$ fer, W., Bastert, J., Chen, W. &
Hube, B. (1999b). Secreted aspartic proteinase (Sap) activity
contributes to tissue damage in a model of human oral candidosis.
Mol Microbiol 34, 169–180.
Schaller, M., Schackert, C., Korting, H. C., Januschke, E. & Hube,
B. (2000). Invasion of Candida albicans correlates with expression

of secreted aspartic proteinases during experimental infection of
human epidermis. J Investig Dermatol 114, 712–717.
Schro$ ppel, K., Sprosser, K., Whiteway, M., Thomas, D. Y.,
Ro$ llinghoff, M. & Csank, C. (2000). Repression of hyphal
proteinase expression by the mitogen-activated protein (MAP)
kinase phosphatase cpp1p of Candida albicans is independent of
the MAP kinase cek1p. Infect Immun 68, 7159–7161.
Sobel, J. D. (1985). Epidemiology and pathogenesis of recurrent
vulvovaginal candidiasis. Am J Obstet Gynecol 152, 924–935.
Smolenski, G., Sullivan, P. A., Cutfield, S. M. & Cutfield, J. F.
(1997). Analysis of secreted aspartic proteinases from Candida
albicans : purification and characterization of individual Sap1,
Sap2 and Sap3 isoenzymes. Microbiology 143, 349–356.
Soll, D. R. (1997). Gene regulation during high-frequency
switching in Candida albicans. Microbiology 143, 279–288.
Staib, F. (1965). Serum-proteins as nitrogen source for yeastlike
fungi. Sabouraudia 4, 187–193.
Staib, P., Kretschmar, M., Nichterlein, T., Ko$ hler, G., Michel, S.,
Hof, H., Hacker, J. & Morschha$ user, J. (1999). Host-induced,
stage-specific virulence gene activation in Candida albicans during
infection. Mol Microbiol 32, 533–546.
Staib, P., Kretschmar, M., Nichterlein, T., Hof, H. & Morschha$ user,
J. (2000). Differential activation of a Candida albicans virulence
gene family during infection. Proc Natl Acad Sci U S A 97,
6102–6107.
Stehr, F., Felk, A., Kretschmar, M., Schaller, M., Schafer, W. &
Hube, B. (2000). Extracellular hydrolytic enzymes and their
relevance during Candida albicans infections. Mycoses 43, Suppl.
2, 17–21.
View publication stats
Candida SAP gene family
Togni, G., Sanglard, D., Quadroni, M., Foundling, S. I. & Monod,
M. (1996). Acid proteinase secreted by Candida tropicalis :
functional analysis of preproregion cleavages in C. tropicalis and
Saccharomyces cerevisiae. Microbiology 142, 493–503.
Tsushima, H. & Mine, H. (1995). Cleavage of human big
endothelin-1 by Candida albicans aspartic proteinase. FEMS
Immunol Med Microbiol 11, 69–72.
Tsushima, H., Mine, H., Kawakami, Y., Hyodoh, F. & Ueki, A.
(1994). Candida albicans aspartic proteinase cleaves and
inactivates human epidermal cysteine proteinase inhibitor,
cystatin A. Microbiology 140, 167–171.
Watts, H. J., Cheah, F. S., Hube, B., Sanglard, D. & Gow, N. A.
(1998). Altered adherence in strains of Candida albicans
harbouring null mutations in secreted aspartic proteinase genes.
FEMS Microbiol Lett 159, 129–135.
White, T. C. & Agabian, N. (1995). Candida albicans secreted
aspartic proteinases : isoenzyme pattern is determined by cell
type, and levels are determined by environmental factors. J
Bacteriol 177, 5215–5221.
White, T. C., Miyasaki, S. H. & Agabian, N. (1993). Three distinct
secreted aspartic proteinases in Candida albicans. J Bacteriol 175,
6126–6133.
Wright, R. J., Carne, A., Hieber, A. D., Lamont, I. L., Emerson,
G. W. & Sullivan, P. A. (1992). A second gene for a secreted aspar-
tate proteinase in Candida albicans. J Bacteriol 174, 7848–7853.
Zaugg, C., Borg-Von Zepelin, M., Reichard, U., Sanglard, D. &
Monod, M. (2001). Secreted aspartic proteinase family of Candida
tropicalis. Infect Immun 69, 405–412.
2005